In recent years recombinant protein/enzyme production for use in industrial processes has become more and more important and it is expected that soon many industrial processes will involve recombinant technologies.
However, the expression of recombinant peptides and proteins is still limited, as large efforts are required in order to obtain the desired peptides and proteins with a native fold, in high amounts and high purity.
Generally, product purification is expensive and especially the final step to 100% purity tends to increase the costs exponentially. Especially proteins present a particular problem in downstream recovery because they are difficult to separate from one another and are liable to degradation and denaturation (Hacking, A. J. (1986) Economic aspects of biotechnology, Cambridge University Press). To some extent protein secretion approaches have been developed, wherein the desired protein is expressed in a host cell and exported to the extracellular space, in order to circumvent laborious protein purification starting from raw lysate. However, many protein secretion pathways involve proteolytic degradation of the transported polypeptide, leading to at least partially fragmented peptide or protein product or incomplete secretion of the peptide or protein of interest, e.g., due to arrest of the polypeptides in the periplasm. Therefore, Type 1 secretion systems (T1SS), which mostly occur in Gram-negative bacteria, are currently under investigation, as they export their cognate substrates in a single step from the cytosol to the extracellular medium without the formation of periplasmic substrate intermediates, which often undergo at least partial periplasmic proteolysis. Among the family of T1 SS the hemolysin (Hly) T1SS described by Bakkes et al. involving HlyA as transport substrate is of particular interest, as it is devoid of any proteolytic activity (Bakkes et al. (2010), J. Biol. Chem.; 285(52):40573-80) and thus does not degrade the secreted peptide or protein of interest. The hemolysin (Hly) T1SS of E. coli consists of the inner membrane protein HlyB, which is an ATP binding cassette (ABC) transporter, the outer membrane protein TolC and the membrane fusion protein HlyD in the inner membrane. The interacting substrate HlyA is exported through the hemolysin secretion system in an ATP dependent manner. Even though large efforts have been made so far, the Hly T1S system is still not industrially used, as the yields of expressed and secreted peptides or proteins are still too low for application in any commercially relevant process.
Particularly, the recombinant production of large quantities of peptides with high purities is still limited, so that the current production methods for peptides still mostly rely on cost intensive chemical synthesis.
There is still need in the art for methods that allow efficient production of a recombinant peptide or protein of interest.